A.

Modes of separation capillary electrophoresis

1. Capillary Zone electrophoresis
2.Capillary iso-electric focusing
3. Micellar electrokinetic Capillary chromatography
4. Capillary electrochromatography
5. Capillary gel electrophoresis

B. Electrophoresis for Bio-Applications

DNA, RNA, and protein

Capillary iso-electric focusing

O
OH
NH3
+

pH

pH
O

O
NH3
+

iso-electric
zwitterionic
In this case, the electroosmotic force is weaker than elctrophoretic force.

Micellar electrokinetic Capillary chromatography

Separation of neutral solute
Psuedo-stationary phase

Advantage: easy to apply

Disadvantage: less selectivity

Capillary electrochromatography
Capillary electrochromatography is an electroosmotically driven
liquid chromatographic technique.

Stationary phase

Mobile phase

Capillary electrochromatography:

yes

yes

Capillary electrophoresis:

no

yes

Charged solutes

Neutral solutes

Capillary electrochromatography:

yes

yes

Capillary electrophoresis:

yes

no

Capillary Electrochromatography: analysis of

polycyclic aromatic hydrocarbones

Stationary phase: 90% 3-m octyldecyl-silica particles; 10% 1-m silica

Partition stationary phase
Stabilization of
Electroosmotic flow
Mobile phase: mixture of acetonitrile and 4mM soldium tetraborate solution
R. N. Zare, et al., Anal. Chem. 1995, 67, 2026

Capillary Electrochromatography: analysis of

polycyclic aromatic hydrocarbones

N = 5.44 (tR/wh)2 = 16 (tR/wb)2

R. N. Zare, et al Anal. Chem. 1995, 67, 2026

5. Capillary gel electrophoresis

---------------

Gel
---------------

----------------------------a. Blocking the solute diffusion caused by Joule heating

b. Size of the channels in the gel gives further selection (entropy effect)

B. Electrophoresis for Bio-Applications

Separation of DNA, RNA, and protein

Gel electrophoresis

Capillary gel electrophoresis

---------------

---------------

Electroosmosis can play a significant role in capillary gel

electrophoretic separation, but not in gel electrophoretic separation.
Both techniques separate solutes by their eletrophoretic mobility.

Separation of DNA and RNA

1
L

Separation of Protein

Size effect and electrophoretic mobility

Further applications
-- an example: DNA sequencing

Polymerizable Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)

One-mer difference

Electrophoresis

Automatic DNA Sequencing

One lane: capillary electrophoresis

G, T, G, and C terminators are labeled

by fore different dyes respectively.

A.Important Concepts:
Electrophoresis
Electrophoretic mobility

Separation

Capillary electrophoresis
Electroosmosis and Electroosmotic flow
Apparent Mobility

Driving force

Separation Efficiency

B. Modes of separation capillary electrophoresis

1. Capillary Zone electrophoresis
2.Capillary iso-electric focusing
3. Micellar electrokinetic Capillary chromatography
4. Capillary electrochromatography
5. Capillary gel electrophoresis

C. Bio-Applications

Separation Sciences
1. Introduction: Fundamentals of Distribution Equilibrium
2. Gas Chromatography (Chapter 2 & 3)
3. Liquid Chromatography (Chapter 4 & 5)
4. Other Analytical Separations (Chapter 6-8)
a. Planar chromatography
b. Supercritical fluid chromatography
c. Electrophoresis
d. Centrifugation
e. Field Flow Fractionation

Homework III (b)

1. What is electrophoresis and electroosmosis?
2. Explain how neutral molecules can be separated by micellar
Electrokinetic capillary chromatography.
3. Compare HPLC and Capillary electrochromatography.
4. Compare capillary gel electrophoresis and gel electrophoresis.
5. The observed behavior of benzyl alcohol (C6H5CH2OH) in
capillary electrophoresis is given below. Explain what happens as
voltage is increased.
Electric field (V/m)
6400
12700
19000
25500
31700
38000

Number of plates
38000
78000
96000
124000
124000
96000